ASSIGNMENT

ANALYTICAL LAB AUTOMATION

CHROMATOGRAPHY

Arranged by:

Adam Prabowo (161110199)

Faculty of Medical Applied Science

Sulaiman Al Rajhi University

KSA,2020
HISTORY1

Chromatography may have had beginnings in 1855 when Runge published his work on
dye separation on paper. Perhaps the real start of chromatographic analysis should be credited to
Coppelsroeder, who presented some of his work in 1861 and Schonbein, who described
separation of substances adsorbed onto filter paper. Fischer and Schmidner were aware of these
works according to their reports which came out in 1892. However, the concept of separations on
columns might be attributed to the work of Reed, who described some his early experiments
using columns in 1893. Day, in 1897, demonstrated the use of columns for separation of
petroleum fractions. A short time later, Engler and Albrecht followed with descriptions of
fractionation by coltecting eluants from a column. There have been other workers during this
time who were practicing “chromatography” before Tswet published his work in the separation
of fragments. The following review presents further development of chromatography which had
a renaissance in the 1930’s. The “rediscovery” of chromatography as it is now known dates from
the reports of separations on columns by Kahn and Lederer. Lederer separated carotenoids from
egg yolk following perusal of Tswett‘s descriptions of chromatography.

1861 1892
1855
Coppelsroeder Fischer and
Runge published dye started Schmidner described
separation paper chromatographic separation of
analysis substance

1930 1897
Tswet published his 1893
Engler and Albrecht
work in the collecting eluants Reed used columns
separation of from a column
fragments
DEFINITION

Chromatography is a mixture separation technique based on differences in the

distribution of the components of the mixture between two phases, namely the stationary phase
(solid or liquid) and the mobile phase (liquid or gas). If the stationary phase is an active solid, the
term adsorption chromatography is known. If the stationary phase is liquid, this technique is
called partition chromatography.

Separation
Technique A Mixture Components

Important Term

1. Polarity
 Shows the separation of positive and negative charge poles of a molecule as a
result of the formation of certain configurations of the constituent atoms
 These molecules can be attracted by other molecules that have polarity
 The degree of separation of these molecules determines their polarity and
attractiveness

Polarity is used as an indicator of:

a. Solvents
b. Adsorbents
c. Solutes

LIKE DISSOLVES LIKE Principles:

• Polar solvents tend to dissolve solutes polar

• Polar adsorbents tend to adsorb solutes polar

2. Partition
 The partitioning process depends on the solubility of the solute in two types of
liquid
 Sensitive to differences in BM solut
 Substances consisting of a series of homologous series are best separated by
partition chromatography.
 For example: the separation of various types of amino acids, fatty acids, sugar
TYPES OF CHROMATOGRAPHY2

Chromatography

Solid Ion Gel

Stationary Phase Exchange

Adsorpstion Ion Exchange Exclusion

Mobile Phase Gas Liduid Liduid Liduid

Plate Column Anion Cation GPC

This division can then be further divided as seen in the following scheme:

1. Gas Chromatography
a. GLC : Gas Liquid Chromatography
b. GSC : Gas Solid Chromatography
2. Liquid Chromatography
a. HPLC : High Performance Liguid Chromatography
b. LLC-PC : Liquid Liquid Chromatography-Paper Chromatography
c. LSC-TLC : Liquid Solid Chromatography-Thin Layer Chromatography
d. Ion Excange
e. Exclusion
 GP : Gel Permeation
 GF : Gel Filtration
Paper Chromatography

Paper chromatography is chromatography that uses pure cellulose paper which has a
great affinity for water or other polar solvents. Paper chromatography is used to separate the
mixture from its substance into its components.

Working Principles of Paper Chromatography

The solvent moves slowly on paper, the components move

at different rates and the mixture is separated based on differences
in color spots.

How to Use Paper Chromatography

1. The paper used is Whatman Paper No.1.

2. The sample is dropped on the paper chromatography baseline.
3. The paper is hung in a container containing solvent and saturated with solvent vapor.
4. Filling air with steam, stopping the evaporation of solvents as well as the movement of
solvents on paper
Column Chromatographic

Column chromatography is the most useful method of separating compounds in a

mixture. Fractionation of solutes occurs as a result of differential migration through a closed tube
of stationary phase, and analytes can be monitored while the separation is in progress. In column
liquid chromatography, the mobile phase is liquid and the stationary phase can be either solid or
liquid supported by an inert solid. A system for low-pressure.

Principles of Column Chromatography

1. Based on absorption of mixed components

with different affinity for stationary phase
surfaces.
2. Absorbent acts as a stationary phase and
the mobile phase is the liquid that flows
carrying mixed components along the
column.
3. Samples that have a large affinity for
absorbent will be selectively held back and
their smallest affinity will follow the flow
of the solvent.

How to use column chromatography

1. The sample is dissolved in a small amount of solvent, poured through the top of the
column and allowed to flow into the adsorbent (absorbent material).
2. Components in the sample are adsorbed quantitatively by the absorbent material in the
form of a narrow band on the top surface of the column.
3. With the addition of solvents continuously, each component will move down through the
column and a ribbon will be formed which each zone contains one kind of component.
4. Each zone that exits the column can be accommodated perfectly before the other zones
exit the column.
Thin-Layer Chromatography3

Thin-layer chromatography (TLC), first described in 1938, has largely replaced paper
chromatography because it is faster, more sensitive, and more reproducible. The resolution in
TLC is greater than in paper chromatography because the particles on the plate are smaller and
more regular than paper fibers. Experimental conditions can be easily varied to achieve
separation and can be scaled up for use in column chromatography, although thin-layer and
column procedures are not necessarily interchangeable, due to differences such as the use of
binders with TLC plates, vapor-phase equilibria in a TLC tank, etc. There are several distinct
advantages to TLC: high sample throughput, low cost, the possibility to analyze several samples
and standards simultaneously, minimal sample preparation, and that a plate may be stored for
later identification and quantification. TLC is applied in many fields, including environmental,
clinical, forensic, pharmaceutical, food, flavors, and cosmetics. Within the food industry, TLC
may be used for quality control. For example, corn and peanuts are tested for
aflatoxins/mycotoxins prior to their processing into corn meal and peanut butter, respectively.
Applications of TLC to the analysis of a variety of compounds, including lipids, carbohydrates,
vitamins, amino acids, and natural pigments, are discussed in reference.

How to use Thin Layer Chromatography

The method of using TLC is almost

the same as the use of paper chromatography,
except that the stationary phase of TLC uses
glass plates / metal / aluminum foil / silica gel
while paper chromatography uses filter paper.
Gas Chromatography

Gas chromatography is a group of chromatographic separation techniques that use gas as

a mobile phase. Because gas is spread to the space around it. hence there is no gas
chromatography in the planar form, all gas chromatography is in the form of a column. The
column for gas chromatography is usually made of stainless metal or glass. In the separation
groove, the sample phase is also gas. Because generally the sample for liquid-phase gas
chromatography, the gas chromatography separation system requires heating. On the other hand,
gas stability is strongly influenced by temperature, so that even though the sample is gas phase, a
heating system to allow temperature control is still needed.

Gas chromatography (GC), including analytical tools. Analysis can be divided into:

1. Qualitative Analysis, namely determining the properties of a component or mixture of

components.
2. Quantity Analysis, which is determining the amount of a component or components in a
mixture.

Gas chromatography is aligned as a method of analysis that can be used to analyze organic
compounds. Based on the stationary phase, there are two types of gas chromatography known
that is :

1. Gas Solid Cromatography, GSC

2. Gas Liquid Cromatography, GLC

In both cases the mobile phase is gas (so they are called gas chromatography) but the
stationary phase is different. Nevertheless both methods have many similarities in how they
work. In GSC we have absorption (absorption) and in GLC we have partition (solution).

GSC operation requires a higher level of expertise than GLC, so it is relatively rarely used.
the cause is GSC based on solid stationary phase, so that the analyte mooring force is based on
physical adsorption. This mooring force is semi-permanent to polar or active molecules so that
the adsorption process is non-linear, consequently the elution pic becomes very tailed. GLC is
relatively easy to operate so it is widely used in all fields of science, and the name GLC is often
abbreviated as Gas Chromatography (GC).
1.1 Gas Solid Cromatography, GSC

Gas Chromatography is a technique for separating a mixture of volatile

substances by passing the gas flow in a stationary phase. The basic work of the GSC
is adsorption (uptake). For this reason, the GSC is very difficult to use repeatedly
with the same results. This is caused by the fact that:

1. The activity of the adsorbent depends on how it is made.

2. Also the activity depends on how it is treated after its manufacture.

Conditions 1 and 2 are very difficult to standardize. These are the things that cause
the low "Reproducibility" of the GSC. Frequently encountered are:

a. The caudate peaks are due to the inhomogeneous active surface of the absorber.
b. Retention time is relatively long.
c. Retention time is very dependent on the number of footage.
d. It is possible that the absorbent can act as an active catalyst

That is why the use of the GSC is very limited, both for compounds that have low
or high boiling points. However there are advantages to GSC when compared to
GLC. The stationary phase cannot be evaporated, because the vapor pressure from the
solid is very low. Until it can use sensitive detectors, because here does not happen
"column bleeding". In recent years the GSC has become more important, after
finding better absorbents. Examples of absorbents are:

1. "Graphite-coal": known as "spheron", the structure is very homogeneous

(compared to diamond): it is used for polar compounds with high boiling
points.
2. "Molecular sieves": these are artificial zeolites (= Na- or CaAI-silicate), with
very small pores in them. The size of the pore diameter is expressed in A°. For
example "Linde", has a size of 3A ° to 13A ° Zeolites are very stable, at
temperatures up to 600 ° C. Molecular sieves absorb only molecules that are
smaller than their porosity, water is absorbed very quickly until it is a very
efficient dryer Molecular sieves have catalyst properties that can be
dehydrator substances (dehydrating agents) For example, zeophytes can
convert alcohol to hydrocarbons / gasoline: R-OH → RH. Before use,
molecular sieves are usually activated by heating them for 12 hours at 150 ° C
while drying H2 gas.
 The use of molecular separation of gases such as hydrocarbons absorbs very
strong hydrocarbons. CO2 and the process cannot be reversed so zeolites are not
used if CO2 is used as a transporting gas.

The current development in GSC is the use of porous polymers such as

PORAPAK and POLYPAK (their trade names) as well as CHROMOSORB.

These absorbents are very suitable for separation:

1. Highly polar compounds such as H20, NH3, R-NH2, R-OH and glycols,
and low fatty acids.
2. Also for like CO2, N2O, O2 as well as others.

The oldest gas chromatography used was the GSC. In 1800 the gases were
purified by absorbing the stationary phase in the form of: alumina (AI203), or
silica gel (Si O, purified sand). Sometimes these absorbents become inactive
when exposed to water.

1.2 Gas-liquid chromatography (GLC)4

Gas-liquid chromatography (GLC) can be used to determine physical constants as

well as for quantitative analyses. For the latter, only peak areas need be accurately
known, but accurate determination of physical constants, such as partition and
activity coefficients, requires that certain corrections be applied to peak position and
width for detector volume. Even in calculating column efficiency such corrections are
important. The equations relating detector concentration to volume of carrier gas are
derived for a model apparatus, which is shown to represent an actual gas-liquid
chromatography apparatus with sufficient accuracy for this purpose. The necessary
corrections for the detector volume within wide limits of retention volume, column
efficiency, and detector volume can be calculated by the use of graphs.

Sensitivity:

GLC is very sensitive. The simplest tool can detect concentrations in 0.01% size
(= 100 ppm). More complicated GLC tools can detect compounds whose
concentrations are only a few specific tools that can now be made with the ability to
detect "parts per billion", up to a picogram range of 10 g. Due to the high sensitivity
of the GLC it only requires a small amount of footage, usually in microliter size.
Liquid Liquid Chromatography (LLC)

LLC is division chromatography in which the partition occurs between the mobile phase
and the stationary phase of both the liquid. In this case the stationary phase must not dissolve in
the mobile phase.

Generally as a stationary phase water is used and as a mobile phase is an organic solvent.
For example in paper chromatography, as the stationary phase is water that is absorbed in the
cellulose fibers from the paper.

Liquid Solid Chromatography (LSC)

LSC is absorption chromatography. As an adsorbent silica gel, alumina, molecular or

porous glass filters are packaged in a column where the mixed components are separated by the
mobile phase. Column chromatography and thin layer chromatography (TLC) are separation
techniques that fall into this class.

High-performance liquid chromatography (HPLC)

High Performance Liquid Chromatography (HPLC) is a chromatographic method that is

capable of separating mixtures of macromolecules, ionic compounds, unstable natural products,
polymeric compounds and polyfunctional groups that have high molecular weight by means of
fractionation absorption, ion absorption or exchange.

In terms of equipment the HPLC system includes column chromatography because the
stationary phase is loaded or packed in the column. And when viewed from the separation
process, HPLC is classified in the adsorption or partition chromatography. HPLC is a very useful
tool in analysis. This section explains how the implementation and use and principles of HPLC
are the same as thin layer chromatography and column chromatography.

Chromatographic separation in HPLC is the result of specific interactions between the

sample molecules with the stationary or mobile phases.

HPLC is also the most widely used chromatography technique, with advantages:

1. Sensitive and easily adjusted for quantitative analysis

2. Suitable for the separation of species that are volatile and species that are easily damaged
by heat
3. Can be used for industry, various fields of science and public purposes such as for the
analysis of amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, drugs,
terpenoids, pesticides, antibiotics, steroids, organic metal species, and various inorganic
compounds.
 HPLC uses a liquid mobile phase, the
stationary phase can be liquid or solid, and can
be divided into using a liquid mobile phase, the
stationary phase can be liquid or solid, and can
be divided into 4 (four) main types, namely:

1. Partition chromatography (for solutes of

small polar compounds but not ionic)

2. Adsorption Chromatography or Padar-liquid

Chromatography (for the separation of non-
polar species, structural isomers, and
classification of compounds such as the
separation of aliphatic hydrocarbons from
aliphatic alcohols)

3. Ion chromatography (for low Mr ionic species)

4. Size-exclusion chromatography or gel chromatography (for solutes with Mr > 1000) which
can be separated again into gel filtration and gel permiation.

Ion-exchange chromatography

This technique uses zeolitas, organic or inorganic resins as ion exchangers. Compounds
which have ions with different affinity for the resin used can be separated. Analysis of amino
acids is common in this way. Other examples are nucleic acids and analysis of inorganic salts.

Exclusion chromatography

In this technique, many porous nonionic gels of the same size are used to separate
mixtures based on differences in molecular size. Small molecules will enter the pores of the gel
while large molecules will pass through the sidelines of the gel more quickly when compared to
molecules that pass through the pores. So the initial elution sequence is the larger molecule, the
medium molecule, and finally the smallest molecule. If as a filter hydrophilic gel (Sephadex) is
used, this technique is called gel filtration chromatography and when using a hydrophobic gel
(polystyrene-divinylbenzene) is called gel permeation chromatography.

Commonly used chromatographic techniques in pharmaceuticals are column

chromatography, paper chromatography, thin layer chromatography, gas chromatography, and
high performance liquid chromatography (high performance liquid chromatography / HPLC).
 REFERENCES

1. Touchstone, J. C. (1993). History of Chromatography. Journal of Liquid

Chromatography, 16(8), 1647–1665. doi:10.1080/10826079308021679 
2. Heftmann E (ed) (2004) Chromatography, 6th edn. Fundamentals and applications of
chromatography and related differential migration methods. Part A: fundamentals and
techniques. Part B: applications. J Chromatogr Library Ser vols 69A and 69B. Elsevier,
Amsterdam
3. Wall PE (2005) Thin-layer chromatography: a modern practical approach. The Royal
Society of Chemistry, Cambridge, UK
4. Johnson, H. W., & Stross, F. H. (1959). Gas-Liquid Chromatography. Analytical
Chemistry, 31(3), 357–365. doi:10.1021/ac60147a010